Coumarin chelates

ABSTRACT

There are described stable fluorescent labels comprising a complex of lanthanide metal and a chelating agent comprising a nucleus which is a triplet sensitizer having a triplet energy greater than that of said lanthanide metal and at least two heteroatom-containing groups which form coordinate complexes with lanthanide metals and a third heteroatom-containing group or heteratom in or appended to the triplet sensitizer. Labeled physiologically active materials useful in specific binding assays such as labeled antigens, heptens, antibodies, hormones and the like comprising the stable fluorescent labels having physiologically active materials adsorbed or bonded thereto are also described.

The present case is a Rule 60 divisional of U.S. Ser. No. 825,693, filedFeb. 3, 1986, now U.S. Pat. No. 4,637,988, which in turn was a Rule 62continuation of U.S. Ser. No. 279,398, filed July 1, 1981, nowabandoned.

FIELD OF THE INVENTION

The present invention relates to novel fluorescent labels and moreparticularly to fluorescent labels useful for the preparation ofspecific binding reagents comprising fluorescent labeled physiologicallyactive materials.

BACKGROUND OF THE INVENTION

In specific binding assays, sensitivity is of prime importance due tothe generally low analyte levels that are measured. Radioimmunoassaysensitivity limits the assay to measurements of concentration of 10⁻¹²M, and more often only in the 10⁻⁸ to 10⁻¹⁰ M range. In addition,radiolabels suffer from the drawbacks of short half life and handlinghazards.

In fluorescence spectroscopy assays, a sample containing a fluorescentspecies to be analyzed is irradiated with light of known spectraldistribution within the excitation spectrum of the target fluorescentspecies. The intensity of the resulting characteristic emission spectrumof the fluorescent target molecules is determined and is related to thenumber of target molecules.

The sensitivity of fluorescence assays, although theoretically veryhigh, is limited by the presence of background fluorescence. Backgroundsignal levels are picked up from competing fluorescent substances, notonly in the sample, but also in materials containing the sample. This isan especially serious problem in quantitative measurements of speciesassociated with samples containing low concentrations of desired targetfluorescent molecules such as found in biological fluids. In manysituations, it is impossible to reduce the background sufficiently (byappropriate filtration and other techniques known in the art) to obtainthe desired sensitivity.

Time resolution offers an independent means of isolating the specificfluorescent signal of interest from nonspecific background fluorescence.Time resolution is possible if the label has much longer-livedfluorescence than the background, and if the system is illuminated by anintermittent light source such that the long-lived label is measureableduring the dark period subsequent to the decay of the short-livedbackground. Such techniques are described in greater detail in GermanOffenlegungsschrift No. 2,628,158 published Dec. 30, 1976.

The long-lived fluorescence (0.1-5 msec) of the aromatic diketonechelates of certain rare-earth metals, for example,europiumbenzoylacetonate and europiumbenzoyltrifluoracetonate, has beenknown for some time. The chelating agent absorbs light and transfers itto the metal ion, which fluoresces. German OLS No. 2,628,158 describesthe use of time resolution in fluorometric immunoassays (FIA) throughthe use of fluorescent labels whose emissions are long-lived as comparedwith those of species which produce background interferences in suchassays. This publication also provides a useful discussion of thetechniques of FIA and its advantage over other immunoassay techniquessuch as radioimmunoassay (RIA).

The fluorescent immunoreagents described in German OLS No. 2,628,158comprise at least one member of the immune system, i.e., an antibody oran antigen, "conjugated" with a rare-earth chelate. Such "conjugation"can be achieved in one of two ways:

(1) first, by labeling, i.e., attaching the rare-earth chelate to theantigen as described in Fluorescent Antibody Techniques and TheirApplication by A. Kawamura, Ed., University Park Press, Baltimore, Md.,1969, and then adding antibody to the conjugated antigen whereby theantibody and antigen join in the usual fashion, or:

(2) by covalent bonding of the antibody to the chelate via a chemicalgroup which binds to both antibodies and the chelates.

The problem with immunoreagents of the type described in German OLS No.2,628,158 is that the fluorescent labeling species, namely, therare-earth chelates, are quenched, i.e., their fluorescence isextinguished, when contacted with water. This problem, hereinafterreferred to as an "aqueous stability" problem, is particularly seriousbecause a principal use for fluorescent labeled immunoreagents is in theassay of aqueous biological liquids such as blood, serum, etc. Ifaqueous stability could be conferred on these materials, they would beuseful as fluorescent labels for these biological liquids, thus allowingincreased fluorescence immunoassay sensitivity by the use of timeresolution of signal from background.

Further, rare-earth chelates previously used for fluorometricmeasurements have had undesirable properties such as a low quantum yieldfor emission, undesirable sensitizer extinction coefficients whichresult in insufficient fluorescence using small quantities of detectablespecies, low λmax which renders the determination subject tointerference from other components in the sample which are usually inthe low λmax range, poor water solubility (most biological fluids areaqueous) and poor stability of the chelate at low concentrations.

SUMMARY OF THE INVENTION

Highly fluorescent compounds have been discovered which are chelates ofa lanthanide metal and a chelating agent comprising a nucleus which is atriplet sensitizer having a triplet energy greater than that of thelanthanide metal and at least two heteroatom-containing groups whichform chelates (coordinate complexes) with lanthanide metals, and a thirdheteroatom-containing group or heteroatom which is in the sensitizer orappended to the sensitizer nucleus. The chelates are water-soluble,stable at low concentrations at pH of 8 or 10, highly sensitive, andhave favorable molar extinction coefficients (10,000-40,000) andfavorable λmax. Accordingly, the present invention provides a class ofhighly efficient, aqueous-stabilized fluorescent labels forphysiologically active materials such as antigens and hormones. Thepresent invention also provides a new class of specific bindingreagents, such as antigens, enzymes, hormones and the like bearing thesehighly useful fluorescent labels.

The reagents are formed by adsorbing or covalently binding thefluorescent labeled antigens, haptens, antibodies, plant lectins,carbohydrates, hormones, enzymes and other such species-specificmaterials.

DESCRIPTION OF PREFERRED EMBODIMENTS

Generally, any lanthanide metal is useful in the chelates describedherein. Examples of lanthanide metals useful herein are europium andterbium and are described by Sinha, S. P., Complexes of Rare Earths,Pergamon Press, 1966.

The lanthanide metal is complexed with a chelating agent comprising anucleus which is a triplet sensitizer having a triplet energy greaterthan that of the lanthanide metal and at least two heteroatom-containinggroups and a third heteroatom-containing group or heteroatom which is inor appended to the triplet sensitizer nucleus, each of said twoheteroatom-containing groups appended to different carbon atoms of thetriplet sensitizer nucleus, said heteroatom-containing groups formingcoordinate complexes with lanthanide metals and said groups beinglocated in said chelating agent such that they and said third heteroatomor heteroatom-containing group are capable of forming a chelatestructure with the lanthanide metal.

The nucleus of the chelating agent is any triplet sensitizer having therequisite triplet energy. Examples of triplet sensitizers useful hereininclude ketones such as benzophenone, propiophenone, Michler's ketone,acetophenone, 1,3,5-triacetylbenzene, isobutyrophenone,1,3-diphenyl-2-propanone, triphenylmethyl phenyl ketone,1,2-dibenzoylbenzene, 4,4'-dichlorobenzophenone, 1,4-diacetylbenzene,9-benzoylfluorene, p-cyanobenzophenone, β-naphthyl phenyl ketone,2-acetonaphthone, α-naphthyl phenyl ketone and 1-acetonaphthone,including α,β-diketones such as biacetyl, benzil and 2,3-pentanedione; aketoaromatic compound such as xanthone, thioxanthone, anthraquinone,α-naphthoflavone, flavone, 5,12-naphthacenequinone and fluorenone; analdehyde such as benzaldehyde, phenylglyoxal, ethyl phenylglyoxalate,2-naphthaldehyde and 1-naphthaldehyde; a linear or fused polycyclicaromatic compound such as fluorene, triphenylene, phenanthrene,naphthalene and pyrene; heterocyclic and aromatic nitrogen-containingcompounds such as carbazole, terpyridines, phenanthroline,triphenylamine, thiazolines, especially 2-organocarbonylthiazolines suchas 2-benzoylmethylene-1-methylnaphtho[1,2-d]thiazoline,2-furoylmethylene-1-methylnaphtho[1,2-d]thiazoline,2-(difuroylmethylene)-1-methylnaphtho[1,2-d]thiazaline,1-methyl-2-thenoylmethylenenaphtho-[1,2-d]thiazoline and2-(dithenoylmethylene)-1-methylnaphtho[1,2-d]thiazoline; thiazolinecompounds as described in U.S. Pat. Nos. 2,732,301 and 4,119,466; andketocoumarins such as described in U.S. Pat. No. 4,147,552.

When the lanthanide metal is europium the triplet energy must be atleast about 47 Kcal, and if the lanthanide metal is terbium the tripletenergy of the nucleus must be at least about 53 Kcal.

The chelating agent also comprises at least two heteroatom-containinggroups which are located on the chelating agent such that they arecapable of forming coordinate bonds with lanthanide metals. Groupscapable of forming coordinate bonds with lanthanide metals includenitrilodiacetate, carboxy, hydroxy, alkoxy, amino, amido, carbonyl andmercapto groups. These groups are appended to the nucleus so as to allowchelation of the gorups with the lanthanide metal.

Preferred chelating agents have the structure: ##STR1## wherein:

Z together with R² represents the atoms necessary to complete asubstituted (such as with a group used to link up the immunoreagent suchas ureylene, thioureylene, carbonylimino or imino linked to animmunoreagent, or a coumarin group) or unsubstituted nucleus which is atriplet sensitizer having a triplet energy greater than that of thelanthanide metal;

R² is selected from the group consisting of a heteroatom and an alkylenegroup having at least one heteroatom therein or a heteroatom orheteroatom-containing group appended thereto; and

R³ and R⁴ are heteroatom-containing groups which are the same ordifferent which will form a coordinate bond with a lanthanide metal; R³and R⁴ being in sufficient proximity to R² to allow chelation of thelanthanide metal to R², and wherein the number of carbon and heteroatomsrepresented by R² is equal to or less than 20.

Z together with R² represents the atoms which complete a substituted orunsubstituted nucleus which is a triplet sensitizer as describedhereinbefore.

R² is a heteroatom such as nitrogen, oxygen, sulfur and selenium or analkylene group having therein at least one heteroatom or a heteroatom orheteroatom-containing group appended thereto. The number of total atomsrepresented by R² is equal to or less than 20. Thus, R² can comprise oneor more heteroatoms. Examples of R² are --NH--, O, S, Se, --N═, ##STR2##(R=H, aryl or alkyl), ##STR3## and a heteroatom or aheteroatom-containing alkylene group of up to 10 carbon atoms. Furtherexamples are carbonyl, dicarbonyl, thiocarbonyl, hydroxymethylene,1,2-dihydroxyethylene and 1,2-dihydroxyvinylene.

R³ and R⁴ are independently heteroatom-containing groups such asdescribed hereinabove and are located in sufficient proximity to R² sothat the lanthanide metal chelates with R². It is preferred that R³ andR⁴ be either individually adjacent to R², or three or less atoms removedfrom R².

In one preferred embodiment, the lanthanide metal is chelated with aphenol having nitrilodiacetate groups substituted in each position orthoto the phenolic hydroxy group. The phenol is unsubstituted orsubstituted with a variety of groups such as alkoxy, alkyl, halogen andcarbonyl and is optionally fused to another aromatic group or to analicyclic or heterocyclic group. Especially preferred are compoundshaving the structure: ##STR4## wherein:

M is hydrogen or a cation such as ammonium or its derivatives such astetramethylammonium, tetraethylammonium and benzyltrimethylammonium oralkali metal such as sodium, lithium, potassium, rubidium and cesium;and

D represents the atoms necessary to complete an aromatic ring. Thisaromatic ring must bear a hydroxyl group as shown above. In addition, itmust bear a carbonyl group such as ##STR5## where alkyl generallycontains up to about 10 carbon atoms such as methyl and ethyl and Ar isaryl such as phenyl; or it must be fused at two of its availablepositions to another aromatic, alicyclic or heterocyclic ring preferablycontaining from about 4 to about 7 carbon atoms such as benzene(substituted or unsubstituted), benzophenone and pyran which bears acarbonyl group to form a coumarin nucleus. Examples of the aromatic ringare phenyl, naphthyl and the like and are optionally substituted in anyof the available positions with alkyl, preferably containing from about1 to about 4 carbon atoms such as methyl, ethyl and propyl, hydroxy andaryl such as phenyl; aldehyde groups such as CHO; benzoyl groups suchas: ##STR6## In especially preferred embodiments, D completes a phenylring with a carbonyl substituent such as a benzoyl substituent or itcompletes a coumarin group. Throughout the specification the terms"alkyl" and "aryl" include substituted alkyl and aryl wherein the arylor alkyl are optionally substituted with groups such as methyl, ethyland propyl.

A preferred embodiment of the invention involves the formation of achelate of a lanthanide metal with a salt or acid having the followingstructure: ##STR7## wherein:

M is hydrogen, ammonium or an alkali metal ion, or any other suitablecation which renders the salt water-soluble.

In another preferred embodiment, the organic compound which complexeswith the lanthanide metal has the following structure: ##STR8## wherein:

R₅ is preferably aroyl such as: ##STR9## and

each R₆ is independently hydrogen or alkyl having from about 1 to about4 carbon atoms such as methyl, ethyl, propyl; aryl or aroyl with orwithout further substitution; alkoxy such as methoxy and propoxy; orhalogen such as bromine or chlorine.

The preferred aroyl R₅ substituent is optionally further substitutedwith aryl or alkyl groups, or with ester, amide, carbamide,thiocarbamide, isocyanate, thiocyanate, halogen or nitrile groups.

Certain other coumarin compounds in which R₅ is appended to the coumarinring by other than an electronegative (i.e., an electron-withdrawing)group are known to fluoresce intensely and are not as useful in thepractice of this invention, as this fluorescence prevents the transferof energy of excitation to the europium or terbium complex withsubsequent fluorescence in the visible portion of the spectrum. Theorganic salt or acid used to form the rare-earth chelate must absorb inthe region of 300-500 nm and must then transfer its excitation energy tothe lanthanide metal which then fluoresces in the visible portion of thespectrum. Other examples of useful complexing compounds include:##STR10##

The complex contains any ratio of lanthanide metal to chelating agent.In preferred embodiments, the mole ratio of lanthanide metal tochelating agent is from about 1:1 to about 2:1.

Preferred complexes have a mole ratio of 1:1.

The chelating agents are prepared by performing a Mannich reactionbetween known compounds of the structure ##STR11## and iminodiaceticacid or esters thereof, and formaldehyde; or by a nucleophilicdisplacement reaction between compounds of the structure ##STR12## whichhave active methylene groups such as bromomethyl or methylenetosylategroups, and nitrilodiacetic esters, and in a subsequent step,hydrolyzing the esters.

Useful complexes include ##STR13##

The lanthanide metal and the chelating agent are easily complexed bymerely mixing an aqueous solution of the chelating agent with alanthanide metal salt in an aqueous solution of pH 7.5-10. Thelanthanide metal salt is any water-soluble salt of the metal such aschloride salts such as TbCl₃.6H₂ O; EuCl₃.6H₂ O.

The chelate is generally prepared in aqueous solution at a pH of between8 and 11 and preferably 8 and 9.

The chelate optionally is mixed with buffers such as phosphate andborate to produce the optimum pH.

The chelate is useful to label a variety of physiologically activematerials by binding said materials to the complex by adsorption or bycovalent bonding. Among the physiologically active materials which arelabeled in this fashion are enzymes and their substrates, antigens,i.e., any substance which is capable, under appropriate conditions, ofreacting specifically in some detectable manner with an antibody,carbohydrate, metabolites, drugs, other pharmacalogical agents and theirreceptors and other binding substances. Specific binding assay reagentsare described in U.S. Pat. Nos. 3,557,555, 3,853,987, 4,108,972 and4,205,058.

Techniques for performing such binding of physiologically activematerials to the complexes are those wellknown in the art and includesimply mixing the materials together.

In specific binding assay methods, a compound having structuralsimilarity to the analyte being determined is bonded to a detectablelabel. The analyte being determined is herein referred to as the ligandand the labeled compound as the ligand analog. Compounds whichspecifically recognize the ligands and ligand analogs and bond to themare referred to as receptors.

In performing one such type of assay, the ligand is placed incompetition with the ligand analog for binding to the receptor. Unknownconcentrations of the ligand are inferred from the measured signal ofthe labeled, ligand analog. The reaction proceeds as follows:

    ligand+(labeled) ligand analog+receptor⃡ligand/receptor+ligand analog/receptor

For illustrative purposes, the discussion which follows describes oneparticular type of specific binding assay technique, a competitivebinding fluorescence imunoassay technique.

This system consists of antigen labeled with a fluorescent label of thepresent invention, unlabeled native antigen (in test sample) andspecific antibody whereby there is competition between the unlabeledantigen and the labeled antigen for binding to the antibody.

The greater the concentration of unlabeled antigen from the test samplein the system, the less the labeled antigen will be bound by theantibody. If the concentration of labeled antigen and antibody is fixedand the only variable is the level of unlabeled antigen, it is possibleto determine the unknown level of unlabeled antigen by physicallyseparating the antigen-antibody complex from the remaining free antigen(both labeled and unlabeled) and comparing the fluorescence of thelabeled antigen, either free or bound, with a standard curve plotting ofthe values given by a range of known amounts of the antigen treated inthe same manner.

A preferred fluorescently labeled specific binding reagent comprises acomplex of a lanthanide metal and a chelating agent having thestructure: ##STR14## wherein:

Z, R², R³ and R⁴ are as described above and L' is a linking group suchas an ester such as ##STR15## amide such as ##STR16## sulfonamide suchas SO₂ NH--, ##STR17## ether such as --O--, --S--, carbonyl such as##STR18## nitrilo such as ═N--, and imino such as --NH--, includingthose groups comprising additional organic linking atoms such as aryleneand thioarylene;

l and m are 0 or 1, n is 1 to 3, and R' is a physiologically activematerial such as an antigen or hormone.

Once prepared as described hereinabove, the fluorescent-labeled,physiologically active species is useful in fluorescent specific bindingassays, particularly those which utilize temporal resolution of thespecific detecting signal to distinguish from background as described inaforementioned German OLS No. 2,628,158. In this time-resolved mode(i.e., temporal resolution), the sample is excited in an intermittentfashion and information is accepted only during the dark cycle when thelong-lived fluorescent label is still emitting strongly but when othersources of fluorescence have decayed. Discontinuous excitation isachieved in a variety of ways, including pulsed laser, mechanicalchopping of a continuous excitation beam and moving the sample in andout of the excitation beam. Moreover, discontinuous excitation has theadvantage of allowing the use of high radiant power without theabsorption of a large amount of energy by the sample, thus diminishingthe probability of sample photodegradation.

Examples of such fluorescent specific binding assay techniques whereinthe specific binding reagents described herein find utility aredescribed in U.S. Pat. Nos. 3,998,943, 4,020,151, 3,939,350, 4,220,450and 3,901,654.

In a preferred embodiment, the specific binding assay is carried out ina dry analytical element such as described in copending U.S. applicationSer. No. 973,669 filed Dec. 27, 1978, by Pierce and Frank. In thisembodiment, the element contains a support and a spreading/reagent layercomprised of polymeric beads, and optionally a registration layer. Insome cases, the spreading layer is separate from the reagent layer. Thespreading, reagent and registration layers optionally comprise thepolymeric bead structure. The polymeric beads of the reagent layer havereceptors such as antibodies adsorbed to their surfaces.

The chelate label of the present invention is placed above, below, or inthe reagent layer in a manner that prevents the specific reaction fromoccurring prior to sample wetting, or it is spotted onto the elementconcurrently with or subsequent to the sample. It is only necessary thatthe labeled ligand analog permeate the element upon wetting subsequentlyto compete with the unknown amount of ligand in the sample in theformation of the ligand-receptor complex. The assay is performed byfluorimetrically determining the amount of free labeled ligand analogpresent or the amount of bound labeled ligand analog-receptor complex.

The following nonlimiting examples will serve better to illustrate thesuccessful practice of the instant invention.

EXAMPLE 1

A complex was formed by mixing equimolar amounts of TbCl₃.6H₂ O in anaqueous solution containing a borate buffer which results in a pH of 9with a compound having the structure: ##STR19## which was prepared bythe method described by G. Schwarzenbach et al, Helv. Chim. Acta, 35,1785 (1952). A bright green emission was shown when the solution wasexcited with a long-wavelength UV lamp (Model UVL-21 Blak Ray® lamphaving a λmax at 366 nm).

EXAMPLE 2

To a stirred solution of 9.9 g (0.05 mole) of p-hydroxybenzophenone and13.7 g (0.1 mole) of iminodiacetic acid in 60 ml of water containing 9 gof sodium hydroxide were added slowly 8.9 g of 37% aqueous formaldehydesolution. The mixture was stirred and refluxed 5 hours, then cooled toroom temperature and brought to pH 2 with 2N hydrochloric acid. Thesolid which formed was collected, washed with water and air-dried. Theproduct was recrystallized from 500 ml of 90 percent ethyl alcohol togive 5.9 g of white-to-pinkish solid in two crops.

Anal. calcd. for C₂₃ H₂₄ N₂ O₁₀.H₂ O: C, 54.5; H, 5.2; N, 5.5. Found: C,54.5; H, 4.8; N, 6.1.

UV spectrum (pH 9 borate buffer) λmax 320 nm, ε1.6×10⁴.

An exactly equimolar mixture of the above compound and EuCl₃.6H₂ O orTbCl₃.6H₂ O in pH 9 borate buffer showed a bright red (Eu⁺³) or green(Tb⁺³) fluorescence when excited with a long-wavelength ultravioletlamp. The emission intensity from the above europium chelate solutionwas examined as a function of concentration on a FarrandSpectrofluorimeter® sold by Farrand Instrument Company. The datadisplayed a linear decrease in the logarithm of the emission as afunction of the logarithm of the concentration from 10⁻⁵ to 10⁻⁹ M ineuropium chelate.

EXAMPLE 3

To a solution of 1.64 g (0.02 mole) of 37 percent aqueous formaldehydein 25 ml of methanol were added 3.22 g (0.02 mole) of dimethyliminodiacetate. The solution was concentrated under reduced pressure ona rotary evaporator. Methanol (25 ml) was added to the residue and thesolution was again concentrated. To the remaining oil were added 2.66 g(0.01 mole) of 3-benzoyl-7-hydroxycoumarin followed by 4 ml ofN-methylmorpholine. The mixture was heated with stirring at 115° C. for3 hr. The mixture was concentrated under reduced pressure on a rotaryevaporator. The resulting thick oil was dissolved in a minimum of CH₂Cl₂ and applied to a dry column of silica gel. The column was elutedwith 1:4 ethyl acetate:CH₂ Cl₂. The first yellow band of monoadduct wasdiscarded. The second yellow band was collected. Removal of the solventunder reduced pressure gave 1.6 g of the desired product as a yellow oilwhich could not be induced to crystallize.

To a solution of 1.5 g (0.0027 mole) of the above tetraester in 20 ml ofacetic acid was added 0.6 g (0.003 mole) of cupric acetate monohydrate,followed by 10 ml of water. The mixture was stirred and refluxed undernitrogen for 2 hours. The reaction was cooled to room temperature andbrought to about pH 2 with hydrochloric acid. With stirring, the mixturewas saturated with hydrogen sulfide gas and allowed to stand 15 minutes.The precipitated cupric sulfide was removed by suction filtrationthrough a diatomaceous-earth pad. The clear orange-brown filtrate wasconcentrated under reduced pressure on a rotary evaporator. The residuewas dissolved in hot ethanol containing 25% water, then allowed to coolfinally at 5° C. for several hours. The solid was collected, washed withwater and dried in vacuo to give 0.3 g of product. UV spectrum (pH 9borate buffer) λmax 396 nm, ε27,000.

Analysis calculated for C₂₆ H₂₄ N₂ O₁₂ : C, 56.1; H, 4.3; N, 5.0. Found:C, 55.6; H, 4.2; N, 4.6.

An exactly equimolar amount of the above compound and EuCl₃.6H₂ O in pH9 borate buffer showed a bright red emission when excited with along-wavelength ultraviolet lamp.

The emission intensity from the above europium chelate solution wasexamined as a function of concentration on a Farrand Spectrofluorimeter.The data displayed a decrease in emission as a function of concentrationfrom 10⁻⁵ to 10⁻¹⁰ M in europium chelate.

λmax emission 593 nm, 614 nm, 652 nm, 701 nm

Emission quantum yield over 560 to 800 nm=4.5%

Emission quantum yield at 614 nm=3.7%

EXAMPLE 4 Preparation of2,4-dihydroxy-3,5-bis[N,N-di(ethoxycarbonylmethyl)aminomethyl]benzaldehyde##STR20##

To 8.2 g (0.1 mole) of 37% aqueous formaldehyde in 50 ml of ethanol wereadded 18.9 g (0.1 mole) of diethyl iminodiacetate. The mixture wasconcentrated under reduced pressure on a rotary evaporator. Anadditional 50 ml of ethanol were added and the mixture againconcentrated to dryness. To the resulting oil were added 6.9 g (0.05mole) of solid 2,4-dihydroxybenzaldehyde. The near mixture was stirredand heated at 120° C. for 3 hours, then used without purification.

The above was repeated using dimethyl iminodiacetate with similarresults.

EXAMPLE 5 Preparation of3-(4-nitrobenzoyl)-7-hydroxy-6,8-bis[N,N-di(carboxymethyl)aminomethyl]coumarin##STR21##

To the above Example 4 crude aldehyde (0.05 mole) were added 11.85 g(0.05 mole) of ethyl 4-nitrobenzoylacetate, followed by 100 ml ofethanol. A solution of 30 mg of acetic acid and 42 mg of piperidine in 1ml of ethanol was added, and the mixture stirred and refluxed for 16hours. After this time, the reaction was concentrated on a rotaryevaporator. The resulting oil was dissolved in a minimum of CH₂ Cl₂ andapplied to a silica gel dry column. The column was eluted with 150:850ethyl acetate:CH₂ Cl₂. The first running colorless impurity and a secondrunning dark-yellow impurity were discarded. The slower-movinglight-yellow product band was collected and the solvent removed underreduced pressure. The resulting oil was analyzed by NMR and massspectroscopy and used directly. Yield, 14.3 g.

To 2.67 g (0.00375 mole) of the above tetraester in 75 ml of acetic acidwas added 1.0 g (0.005 mole) of cupric acetate monohydrate, followed by25 ml of water. The mixture was stirred and refluxed under nitrogen for2 hours. The reaction was cooled to room temperature and brought toabout pH 2 with hydrochloric acid. An excess of hydrogen sulfide gas wasthen passed into the stirred solution, and the mixture was allowed tostand 30 minutes. The precipitated cupric sulfide was removed by suctionfiltration through a diatomaceous-earth pad. The filtrate wasconcentrated to dryness under vacuum on a rotary evaporator. The residuewas triturated with 50 ml of water. The solid was collected, washed wellwith water and dried. Yield, 1.2 g. A sample was dissolved in hot 50%aqueous ethanol. The mixture was concentrated under reduced pressureuntil solid formed. the solid was collected, washed with cold water anddried.

Anal. calcd. for C₂₆ H₂₃ N₃ O₁₄.H₂ O: C, 50.4; H, 4.1; N, 6.8. Found: C,50.8; H, 4.1; N, 6.4.

Catalytic reduction of the above chelate in aqueous sodium bicarbonatesolution gave the corresponding amino compound.

EXAMPLE 6

A stock solution containing 10⁻⁴ M concentration of a europium chelatedescribed in Example 3 above was diluted with borate buffer (pH 8.5) toconcentrations shown in Table I. Ten-microliter aliquots of eachconcentration were spotted onto analytical elements prepared in thefollowing manner:

A Lexan® polymer (available from General Electric Company) film supportwas coated with a microbead layer comprised ofpoly(styrene-co-methacrylic acid) (weight ratio 98:2) (75.0 g/m²), whichhad been adsorbed with ovalbumin, carboxymethyl cellulose (0.19 g/m²),Zonyl FSN® (a nonionic fluorosurfactant obtained from duPont), 0.05%based on total melt weight, normal rabbit serum (0.83 g/m²),poly(n-butyl acrylate-co-styrene-co-2-acrylamido-2-methyl propanesulfonic acid) (weight ratio 70:20:10) (2.25 g/m²) and H₃ BO₃.KCl bufferat pH 8.5.

The elements were then evaluated, using a fluorimeter having a Wrattan18A filter, at Excitation₃₀₀₋₄₀₀ nm and Emmission ₆₂₀ nm, at a pH of 8and a pH of 9.18.

The results shown in the table below illustrate that the fluorescencesignal obtained is a function of the concentration of the europiumchelate in the sample. The background fluorescence was 50 mV.

                  TABLE I                                                         ______________________________________                                        Concentration of       Fluorescence at                                        Eu Chelate      pH     Em.sub.620 nm                                          ______________________________________                                        10.sup.-8       8      80-100      mV                                         10.sup.-7       8      400-450     mV                                         10.sup.-6       8      4500        mV                                         10.sup.-5       8      40          V                                          10.sup.-8       9.18   60          mV                                         10.sup.-7       9.18   250         mV                                         10.sup.-6       9.18   2.1         V                                          10.sup.-5       9.18   20          V                                          ______________________________________                                    

EXAMPLE 7

A terbium compound prepared as described in Example 3 was tested in themanner of Example 6. The results are shown in Table II with thefluorescence measured at 550 nm and given in μA.

                  TABLE II                                                        ______________________________________                                        Concentration of       Fluorescence at                                        Tb Chelate (M)  pH     Em.sub.550 nm (μA)                                  ______________________________________                                        10.sup.-5       8.0    11.1                                                   10.sup.-6       8.0    1.41                                                   10.sup.-7       8.0    0.27                                                   10.sup.-8       8.0    0.04                                                   10.sup.-9       8.0    0.05                                                   ______________________________________                                    

EXAMPLE 8 Complex of europium and3,5-bis[N,N-bis(carboxymethyl)aminomethyl]-4'-{N'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-methoxycarbonylphenethyl]thioureido}-4-methoxybenzophenone

The synthetic scheme for the preparation of the chelating agent is asfollows: ##STR22##

Preparation of 3,5-dimethyl-4-hydroxy-4'-nitrobenzophenone (1)

To a stirring solution of p-nitrobenzoyl chloride (76.1 g, 0.410 mol) in250 mL nitrobenzene were added 82 g of AlCl₃ (0.62 mol). To this mixturea solution of 50 grams of 2,6-dimethylphenol (0.41 mole) in 250 mLnitrobenzenewas added dropwise over a period of 45 minutes, and theresulting mixture was stirred 16 hours at room temperature. The reactionwas poured into 2L of 3% HCl and ice and extracted 3×1L with Et₂ O(i.e., three times with 1L each time of diethyl ether). The combinedethereal layers were washed with 1L of saturated NaHCO₃, then extracted2×1L with 10% NaOH. The combined basic extracts were acidified withconcentrated HCl to give a white precipitate; filtration followed byrecrystallization from CH₃ OH/H₂ O gave 37 g (33%) of white powderconsistent with the desired product by TLC, IR, mass spectroscopy, NMRand elemental analysis, m.p. 182.5-184.5.

Anal. Calcd. for C₁₅ H₁₃ NO₄.H₂ O: C, 62.27; H, 5.24; N, 4.84. Found: C,62.34; H, 5.31; N, 4.76.

Preparation of 3,5-dimethyl-4-methoxy-4'-nitrobenzophenone (2)

A solution containing compound l (15.0 g, 55.4 mmol), K₂ CO₃ (20.0 g,0.145 mol) and CH₃ I (30 mL, 0.48 mol) in 250 mL acetone was refluxedfor 6 hours. The solvent was evaporated and the residue partitionedbetween CH₂ Cl₂ and H₂ O. The organic layer was dried over Na₂ SO₄,filtered and evaporated to leave a yellow-white powder.Recrystallization from CH₃ OH/pet ether at -20° C. gave 14.9 g of whitecrystals (94%). The material was characterized by NMR, TLC, massspectroscopy, IR and elemental analysis, m.p. 131°-132.5° C.

Anal. Calcd. for C₁₈ H₁₅ NO₄ : C, 67.35; H, 5.31; N, 4.91. Found: C,67.54; H, 5.38; N, 4.87.

Preparation of 3,5-bisbromomethyl-4-methoxy-4'-nitrobenzophenone (3)

A mixture of compound 2 (4.0 g, 14 mmol) and n-bromosuccinimide (5.0 g,28 mmol) was refluxed in 250 mL CCl₄ with ca. 50 mg dibenzoyl peroxideas a radical initiator for 1 hr under N₂. The reaction was cooled toroom temperature and 100 mL CH₂ Cl₂ were added. Filtration followed byextraction of the filtrate with aqueous sodium thiosulfate, drying theorganic layer over Na₂ SO₄, filtration and solvent removal left a whitepowder. This powder was triturated 3×50 mL with CH₂ CH₂ O to leave 5.7 gof white powder which contained 3 components by TLC. NMR and massspectroscopy confirmed the presence of impurities, but the bulk of thematerial was the desired product.

Preparation of3,5-bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-4-methoxy-4'-nitrobenzophenone(4)

A solution of compound 3 (2.0 g, 4.5 mmol) and di-tert-butyliminodiacetate (2.2 g, 9.0 mmol) was stirred in 200 mL THF for 60 hr atroom temperature. The solvent was removed and the remaining yellow oilwas partitioned between CH₂ Cl₂ and cold aqueous K₂ CO₃ made from highlypurified water. The organic layer was dried over Na₂ SO₄, filtered andevaporated to leave 4.5 g of yellow oil. A preparative gel permeationcolumn with CH₂ Cl₂ as the eluent was used to separate the desiredproduct from starting materials and monoadduct. The resulting yellow oil(1.5 g, 43%) could not be induced to crystallize and was characterizedby TLC, field desorption mass spectroscopy, NMR and IR.

Preparation of4'-amino-3,5-bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-4-methoxybenzophenone(5)

Nitrotetraester 4 (3.0 g, 3.9 mmol) was reduced in 50 mL CH₃ OH with 100mg 10% Pd/C in a Parr shaker with an initial hydrogen pressure of 50 psifor 2.5 hours. TLC indicated quantitative reduction. The mixture wasfiltered through diatomaceous earth and evaporated to leave a yellow oilwhich was purified by gel permeation chromatography by the method usedfor compound 4. The yellow glass thus obtained had the correct NMR, IRand field desorption mass spectroscopic behavior.

Preparation of3,5-bis[N,N-bis-t-butoxycarbonylmethyl)aminomethyl]-4'-{N'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-methoxycarbonylphenethyl]thioureido}-4-methoxybenzophenone(6)

Tetraester amine 5 (1.7 g, 2.3 mmol) together with triethylamine (1.28mL, 9.2 mmol) was dissolved in 40 mL dry tetrahydrofuran (THF), followedby the dropwise addition of thiophosgene (0.175 mL, 2.3 mmol) in 10 mLdry THF. The reaction was allowed to proceed for 2 hours, after whichthe solvent was removed to yield a yellow-orange oil. The oil wasdissolved in 60 mL dry N,N-dimethylformamide (DMF) and a solution ofL-thyroxine methyl ester hydrochloride (1.9 g, 2.3 mmol) andtriethylamine (0.32 mL, 2.3 mmol) in 20 mL DMF was added in one portion.The reaction was stirred 1 hour under N₂, then poured into 350 mL H₂ O.Extraction with ether followed by the ether layer being successivelywashed with three 300-mL portions of purified water, dried over Na₂ SO₄,filtered and evaporated gave 3.1 g of orange-white foam. This materialwas purified by gel permeation chromatography to give 1.3 g of thedesired product as an orange-white foam. The product was furthercharacterized by TLC, NMR and field desorption mass spectroscopy.

Preparation and evaluation of3,5-bis[N,N-bis(carboxymethyl)aminomethyl]-4'-{N'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-methoxycarbonylphenethyl]thioureido}-4-methoxybenzophenone(7 )

Tetraester 6 (0.5 g, 0.3 mmol) was dissolved in 15 mL CF₃ CO₂ H andstirred for 16 hours at room temperature with a drying tube attached tothe reaction flask. The solvent was removed in vacuo to leave an orangefoam. Trituration of the foam with CH₂ CL₂ produced a yellow-orangepowder in quantitative yield. The field desorption mass spectrum shows aparent ion at m/e 1350 and the IR is consistent with the product.

Anal. Calc'd for C₄₁ H₃₈ I₄ N₄ O₁₄ S: C, 36.5; H, 2.8; N, 4.1; S, 2.4.Found: C, 36.4; H, 2.8; N, 3.7; S, 2.7.

One equivalent of the above compound and two equivalents of EuCl₃.6H₂ Oin pH 8.5 borate buffer were weakly fluorescent under long-wavelength UVlight giving the characteristic red emission. A linear relationshipbetween fluorescence intensity and chelate concentration wasdemonstrated between 10⁻⁵ and 10⁻⁷ M with the Varian SF330Spectrofluorimeter® and λex=320 nm, λem=614 nm. One equivalent ofcompound 7 and two equivalents of TbCl₃.6H₂ O in pH 8.5 borate bufferwere strongly fluorescent under long-wavelength UV light. This chelatealso had a linear relationship between fluorescence intensity andchelate concentration between 10⁻⁵ M and 5×10⁻⁸ M.

Cross reactivity is a reflection of how well an antibody recognizes thelabeled antigen as compared with unlabeled antigen. The cross reactivityof the europium chelate of the antigen as determined by known techniqueswas 75% vs. radiolabeled thyroxine and throxine antibody.

EXAMPLE 9 Complex of europium with3,5-bis[N,N-bis(carboxymethyl)aminomethyl]-4-hydroxy-3'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-ethoxycarbonylphenethyl]benzophenone

The synthetic scheme for the preparation of the chelating agent of thisexample is as follows: ##STR23##

3-Carboxy-4'-hydroxybenzophenone (16)

This compound was prepared by the method described in U.S. Pat. No.3,531,435 (Chem. Abstracts 74, 4283V (1971)).

3'-Carboxy-3,5-bis(morpholinomethyl)-4-hydroxybenzophenone (17)

A mixture of 5.22 g (0.06 moles) of morpholine and 1.8 g (9.06 moles) ofparaformaldehyde in 50 mL of isobutyl alcohol was refluxed undernitrogen until a clear solution was obtained. To this solution wereadded 4.8 g (0.02 moles) of 3-carboxy-4'-hydroxybenzophenone (16) andthe refluxing was continued for 3 hours. The solution was evaporatedunder reduced pressure and the gummy residue was stirred several timeswith ether, giving 9.5 g of solid which was not purified.

4-Acetoxy-3,5-bis(acetoxymethyl)-3'-carboxybenzophenone (18)

A mixture of 9.5 g of 17 and 75 mL of acetic anhydride was refluxed for24 hours and the excess acetic anhydride was removed under vacuum. Theresidue was stirred with water and the solid was collected and dried;yield 8.5 g. Thin-layer chromatography (silica gel; 1% methanol inmethylene chloride) shows about 10% of the faster movingmonoacetoxymethyl derivative is present in the 18.

4-Acetoxy-3,5-bisbromomethyl-3'-carboxybenzophenone (19)

A solution of 2 g of 18, 20 mL of methylene chloride and 5 mL of 31%hydrobromic acid in acetic acid was stirred overnight, 3 mL of aceticanhydride were added and the solution was evaporated to dryness. Theresidue was chromatographed on silica gel eluting with 1:1 CH₃ CO₂ C₂ H₅/CH₂ Cl₂ using the method of Still (W. C. Still, M. Kahn and A. Mitra, JOrg Chem, 43, 2923 (1978)), giving 0.56 g of pure 19 as determined byNMR.

4-Acetoxy-3,5-bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-3'-carboxybenzophenone(20)

A solution of 460 mg (1.07 mmol) of 19, 524 mg (2.14 mmol) of tert-butyliminodiacetate, and 216 mg (2.14 mmol) of triethylamine in 15 mL of drytetrahydrofuran was stirred under argon for 2 days. The reaction mixturewas filtered to remove triethylamine hydrobromide. The filtrate wasevaporated to dryness, giving 900 mg of 20.

4-Acetoxy-3,5-bis[N,N-bis(t-butoxycarbonylmethyl)aminomethyl]-3'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-methoxycarbonylphenethyl]benzophenone(21)

A mixture of 900 mg of 20 and 0.265 g (1.07 mmol) of1-carbethoxy-2-ethoxy-1,2-dihydroquinoline (EEDQ) in 20 mL of drytetrahydrofuran was stirred for 30 minutes and 0.85 g (1.07 mmol) of themethyl ester of thyroxine was added. The mixture was stirred overnight.The reaction mixture was purified by gel permeation chromatography usingtetrahydrofuran as the solvent, giving 630 mg of material which showedan NMR spectrum which was consistent with structure 21.

3,5-bis[N,N-bis(carboxymethyl)aminomethyl]-4-hydroxy-3'-[4-(4-hydroxy-3,5-diiodophenoxy)-3,5-diiodo-β-methoxycarbonylphenethyl]benzophenone(22)

A solution of 630 mg of 21 in 5 mL of trifluoroacetic acid was stirredovernight, diluted with water and the solid which separated wascollected and dried; yield 550 mg. The field desorption mass spectrumshowed 22 (m/e1305) is present, as well as some material in which themethyl ester of the thyroxine has been hydrolyzed to the acid.

One equivalent of 22 plus two 2-equivalents of EuCL₃.6H₂ O weremoderately fluorescent when dissolved in pH 8.5 borate buffer andexamined under long-wavelength UV light. A linear plot of fluorescenceintensity vs chelate concentration was generated between 10⁻⁵ and 10⁻⁷ Mfor this compound. All fluorescence measurements were taken with aVarian SF330 Spectrofluorimeter®.

The europium chelate of 22 had a cross reactivity of 80% vs radiolabeledthyroxine and thyroxine antibody.

EXAMPLES 10-16

Europium and terbium complexes with the following chelating agents wereprepared as in Example 9 using EuCl₃.6H₂ O and TbCl₃.6H₂ O in boratebuffer:

    __________________________________________________________________________    Example                                                                             Chelating Agent                                                         __________________________________________________________________________    10                                                                                   ##STR24##                                                              11                                                                                   ##STR25##                                                              12                                                                                   ##STR26##                                                              13                                                                                   ##STR27##                                                              14                                                                                   ##STR28##                                                              15                                                                                   ##STR29##                                                              16                                                                                   ##STR30##                                                              Control A                                                                            ##STR31##                                                              Control B                                                                            ##STR32##                                                              Control C                                                                            ##STR33##                                                              __________________________________________________________________________

The complexes of Examples 10-16 were fluorescent and those of ControlsA, B and C were not fluorescent. Complexes of Controls A, B and C werefurther tested in glycine acetate buffer, phosphate buffer and sodiumbicarbonate buffer and were not fluorescent with either EuCl₃.6H₂ O orTbCl₃.6H₂ O.

The invention has been described in detail with particular reference tocertain preferred embodiments thereof, but it will be understood thatvariations and modifications can be effected within the spirit and scopeof the invention.

What is claimed is:
 1. A chelate having the formula: ##STR34## wherein Mis hydrogen or a cation.